Synthesis of n-5-methyltetrahydrohomofolic acid and related reduced derivatives of homofolic acid

ABSTRACT

The preparation of N5-methyltetrahydrohomofolic acid and alkali metal salts thereof is from homofolic acid starting material (HFA) by catalytic reduction with platinum oxide in the dark under hydrogen at a slight overpressure to produce tetrahydrohomofolate (THHF), then immediately reacting this product with formaldehyde to produce 5,11methylenetetrahydrohomofolate and reducing this methylene intermediate without isolation with sodium borohydride to give the desired 5-methyltetrahydrohomofolate in the disodium salt form. The 5-methyl product is recovered by precipitating side products at a pH of about 3.8 with acetic acid and refrigerating the product filtrate overnight at about 3*-10*C. The filtrate containing the product 5-methyltetrahydrohomofolate (5-MeTHHF) is treated with charcoal, concentrated to an oil and the 5-methyl product is precipitated with absolute ethanol. The 5,11methylenetetrahydrofolate may be separately recovered and in a second reaction an additional product known as the 5,11-methenyl is formed by reaction with formic acid. These products show increased stability over folate analogues and show biological promise in the treatment of cancer and leukemia as antifolates and specifically against L1210-FR8 tumor in mice.

United States Patent [1 Knott et al.

[451 Mar. 11, 1975 1 1 SYNTHESIS OF N- -METI-IYLTETRAIIYDROHOMOFOLICACID AND RELATED REDUCED DERIVATIVES OF I-IOMOFOLIC ACID [75] Inventors:Roger L. Knott; Alison Taunton-Rigby, both of Waltham, Mass.

[73] Assignee: The United States of America as represented by theSecretary of the Department of Health Education and Welfare, Washington,DC.

[22 Filed: June 16, 1972 211 Appl. No.: 263,559

OTHER PU BLlCATlONS Goodman et 211., Pharmacological Basis ofTherapeutics 3rd Ed. (1965), MacMillan Co., N.Y., p. 1429. Gupta et al.,Archiv. Biochem. and Biophys, 120, 712-718, (1967).

Primary Examiner-Donald G. Daus Assistant Examiner-Raymond V. Rush [57]ABSTRACT The preparation of N -methyltetrahydrohomofolic acid and alkalimetal salts thereof is from homofolic acid starting material (HFA) bycatalytic reduction with platinum oxide in the dark under hydrogen at aslight overpressure to produce tetrahydrohomofolate (THHF), thenimmediately reacting this product with formaldehyde to produce 5,] 1-methylenetetrahydrohomofolate and reducing this methylene intermediatewithout isolation with sodium borohydride to give the desiredS-methyltetrahydrohomofolate in the disodium salt form. The 5- methylproduct is recovered by precipitating side products at a pH of about 3.8with acetic acid and refrigerating the product filtrate overnight atabout 310C. The filtrate containing the productS-methyltetrahydrohomofolate (S-MeTHHF) is treated with charcoal,concentrated to an oil and the S-methyl product is precipitated withabsolute ethanol. The 5,1- l-methylenetetrahydrofolate may be separatelyrecovered and in a second reaction an additional product known as the5,1l-methenyl is formed by reaction with formic acid. These productsshow increased stability over folate analogues and show biologicalpromise in the treatment of cancer and leukemiaas antifolates andspecifically against Ll2lO-FR8 tumor in mice.

2 Claims, No Drawings SYNTHESIS OF N-S-METHYLTETRAHYDROHOMOFOLIC ACIDAND RELATED REDUCED DERIVATIVES OF HOMOFOLIC ACID Also reproduced is thestructure analogous to the present compounds.

*Broken lines indicate the N and/or N site of attachment of variousl-carbon units for which THFA acts as a carrier.

in the patent art the production of tetrahydrohomofolic acid salts isset out in:

U.S. Pat. No. 3,468,886 Mosher et a1. (USA-HEW) U.S. Pat. No. 3,637,695Kim et a1. (USA-I-1EW) The folic acid molecule reproduced below isgenerally viewed as consisting of three segments. The first segment is apteridine moity which is a fused diazine with a pyrimidine A ring and a1,4 diazine B ring. A methylene bridge at C connects the first andsecond segments. The second segment is a paraaminobenzoic fraction, andthe third is a glutamic acid residue.

Para-amtnobcnzoylglutemlc Acid ltd-Substituted Ptcrlu Ptcroic Acid *Sitcof conjugation of extra glutamate rcsidue(s) of pteroyl (ii-,trl-Ohnpta-glutumutc.

Folio Acid (ltcroylglutumic Acid) 5,6,7 ,B-Tetrahydrofolic Acid (THFA)(FH4)(R=II) Homofolic acid differs from folic acid in being an analoguewhere an additional methylene or CH group is inserted between C and N ofthe basic folic molecule. Tetrahydrofolic acid is a derivative of folicacid wherein at 5, 6, 7, and 8 positions are hydrogenated.

The most important folic acid derivatives which have been isolated fromliving systems are S-methyl, 5,10- methenyl, and5,lO-methylenetetrahydrofolic acids. The homofolic acid derivativesanalogous to these compounds are, therefore, the most likely candidatesas synthesis for folic acid antagonists to compete with folic and affectits single carbon unit transfer function.

The production of S-methyltetrahydrofolate in the literature dates atleast from 1963 when W. Sakami in Biochem. Prep. 10, 103 (1963) produced5,10- methylenetetrahydrofolate by reacting tetrahydrofolic acid withformaldehyde and subsequently reduced the methylenetetrahydrofolate withpotassium borohydride. The crude 5-methyltetrahydrofolate (MeTHF) formedin this procedure was purified by gradient elution chromotography onDEAE cellulose. V. S. Gupta and F. M. Huennekens in Arch. Biochem.Biophys., 120, 712-718 (1967) followed a similar procedure with thetetrahydrofolate derivatives which also involve condensation offormaldehyde with tetrahydrofolate to yield5,10-methylenetetrahydrofolate followed by reductive cleavage with thelatter of borohydride. Gupta et al found that little or no cleavageoccurred on the N side of the methylene bridge and thus the productionof the S-methyl variety was practically exclusive.

The present invention differs from the art noted above in .thathomofolic acid is utilized as a startingmaterial instead of folic acidand in the procedure following there are several important differencesdesigned to obviate the difficulties encountered by the fragility oftetrahydrohomofolic acid (THHF).

In the present procedure homofolic acid (NSC 79249) is reducedcatalytically in the dark using a platinum oxide catalyst. The catalystis removed under an inert gas such as argon and the resultingtetrahydrohomofolate which is obtained in aqueous solution isimmediately reacted with formaldehyde without light to produce theintermediate 5,1 lmethylenetetrahydrohomofolate. This intermediate,again without isolation, is reduced with an alkali metal borohydridesuch as sodium borohydride to give the desired product,5-methyltetrahydrohomofolic acid in of 5,6,7,8- tetrahydrofolic acid andcompounds in the folic series the disodium salt form. The procedure thusfar emphasizes a rapid sequential process differing from the stepwiseprocess of the prior art.

By use of acetic acid in place of hydrochloric acid and adjusting the pHto 3.8, inorganic salts such as sodium acetate and boric acid arecleanly separated with alcohol (i.e., ethanol) in which they are solublebut from which the desired product, S-methyl THHF, precipitatesessentially quantitatively. The preferred pro-' cedures call for anovernight refrigeration of the impure solution at 3-10C followed byfiltration of impurities. The product in solution may be treated withcharcoal, concentrated to an oil, and precipitated with ethanol(absolute). The yields obtained after drying are in the area of 68percent.

This procedure of separating the sensitive S-methyl THHF producteliminates the time-consuming DEAE column chromatography step and basedon prior procedures with the folate increasead the final yield byapproximately a factor of 5.

A more specific preparation and summary of structure analysis of N-methyl THHF and the intermediate compounds are set out below.

N -methyltetrahydrohomofolic acid is prepared by a general modus asnoted above in the methods of Sakami and of Gupta and Huennekens, ante,for the preparation of the folate compounds. The present synthesisinvolves the condensation of formaldehyde with tetrahydrohomofolate toyield the N N- methylenetetrahydrohomofolate followed by reductivecleavage of the latter with borohydride. As noted in the folate series,this cleavage is highly selective and little or no cleavage occurs onthe N side of the methylene bridge. The starting material,tetrahydrohomofolic acid, was prepared by catalytic hydrogenation ofhomofolic acid at a slight overpressure with platinum oxide. Thecatalyst was removed by filtration and formaldehyde added immediately.After heating to 55C a solution of sodium borohydride was added and themixture incubated at 55C for 2 hours. For structure purposes, theresulting reaction mixture was chromatographed on a DEAE-cellulosecolumn using an ammonium acetate gradient. N -methyltetrahydrohomofolatewas identified as the main peak and isolated by precipitation withabsolute ethanol as a white powder in 46 percent yield. The product wascharacterized by elemental analysis, paper and thin layer chromatographyand absorption spectra at pH 1, 7 and 13.

The cyclic compound N ,N"- methylenetetrahydrohomofolate which is anintermediate in the preparation of N -methyltetrahydrohomofolate wasprepared and isolated as follows. Homofolic acid was reduced totetrahydrohomofolic acid as above by catalytic hydrogenation,formaldehyde added immediately, and the mixture incubated at 55C for 30minutes. The reaction mixture was then chromatographed on DEAE celluloseusing a gradient of trietylammonium bicarbonate (TEAB) containing 1%formaldehyde and 0.5% mercaptoethanol. Again the product wasprecipitated with ethanol as a white powder. Char acterization was byelemental analysis, absorption spectra at different pH s and paper andthin layer chromatography. Reduction with sodium borohydride gave themain product N -methyltetrahydrohomofolic acid.

N ,N -methenyltetrahydrohomofolic acid is produced by the reation ofTHHF with formic acid to give several products depending on reactionconditions where the main product, isolated by DEAE cellulosechromatography, was shown to be N ,N-methenyltetrahydrohomofolic acid ascharacterized by paper and thin layer chromatography and by absorptionspectra.

The tetrahydrohomofolic acid derivatives of the present invention havebeen shown to have substantial antileukernia activity against Ll2l0-FR8tumor in mice and the advantage therapeutically is enhanced by the factthat these compounds show greater stability towards oxidation than theparent compound. The activity of the compound is believed at least inpart to have the function as an inhibitor of thymidylate synthetase.Each of these compounds may be prepared as the free acid or preferablyas an alkali metal disalt of the gluta' mate fraction, such as disodiumsalt. The product compounds exist in the form of racemates due to thepresence of an asymmetric carbon atom at the 6 position. Thus, thestereochemical designation is (d,l).

EXAMPLE 1 Preparation of S-Methyltetrahydrohomofolic Acid (Sodium Salt)NSC-139490 Platinum oxide, 3.0 g, was washed with four 20 ml portions ofwater and was hydrogenated in 130 ml of water at 4 cm overpressure,while stirring vigorously at room temperature. Reduction was complete in2 hours and 50 minutes with absorption of 410 ml of hydrogen.

Homofolic acid (NSC-79249), 5.5 g, was suspended in 200 ml of water and22 ml of 1 M sodium hydroxide was added dropwise (to pH 7.0) over aperiod of one hour while stirring with exclusion of light. The stirringwas continued for an additional hour and thus obtained yellow solutionwas injected into the hydrogenation vessel. Hydrogenation at 4 cmoverpressure was complete in 4 hours and 50 minutes with absorption of590 ml of hydrogen. The reaction mixture was filtered under argonthrough a glass sintered funnel and the filtrate was transferred into a2 liter 3-neck flask which was flushed with argon.

Formaldehyde solution (37%), 2.9 ml, was added, the system was flushedwith argon and the reaction mixture was heated to 55C while stirringunder argon with exclusion of light. ml of a 1.0 M sodium borohydridesolution was added rapidly and the reaction mixture .was subsequentlyincubated at 55C for 2 hours, at which time the evolution of hydrogenhas practically stopped.

The reaction mixture was cooled to room temperature, 8 ml ofmercaptoethanol was added and thus obtained a light yellow-coloredsolution was adjusted to pH 3.8 with glacial acetic acid on a pH meter.The resulting cloudy solution was refrigerated at 3C overnight.

A beige-colored precipitate, containing the impurities, was collected ona Buchner Funnel and the pale straw-colored filtrate was stirred with500 mg of Norit A for 20 minutes and was filtered. Evaporation atreduced pressure gave a viscous oil. The oily residue was thoroughlytriturated with 900 ml of cold ethanol and thus obtained suspension of awhite, solid material which was refrigerated for one hour and thenstirred for one hour while cooling in ice. The white, fluffy precipitatewas collected on a Buchner Funnel, washed repeatedly with cold ethanol,dried for 5 hours on high vacuo and was stored in vacuo.

The product was consistently in accord with the accepted criteria forpurity.

Tlc; cellulose, solvent system; 0.1 M phosphate buffer pH 7, 1%mercaptoethanol Rf 0.80 quenching Analytical data Calculated C, .25; N,17.0; Na, 8.3 Found C, 8

45.5; H, 5 45.4; H, 4. N, 17.7; Na, 8.4

EXAMPLE 11 Preparation of 5,11-Methylenetetrahydrohomofolic Acid 3.2 gof PtO was washed four times with water and hydrogenated with theabsorption of 595 ml of H in 3 hours minutes. 5.5 g homofolic acid washydrogenated with absorption of 600 ml of H in 4 hours 10 minutesutilizing the reduced platinium catalyst. The product was filtered underargon and 1.6 ml of formaldehyde was added. The product was lyophillizedand stored in a deep freeze.

The next day the product (in solution in water) was incubated for 6hours at 55C with an additional 16.5 ml of formaldehyde under argon. Thereaction mixture was then cooled to room temperature and stirred forminutes with 300 mg Norit A. 30 ml of acetic acid was added whilestirring and the pH was adjusted to about 3.5. An off-white precipitatecollected which was washed with alcohol, dried, and stored in vacuo. Theproduct, 5,1 l-methylene THHF, sodium salt, had the following physicalcharacteristics.

uv-spectra, mu; Amax Amin Ash H l 278 255 307 Tlc; cellulose, solventsystem; 0.1 M phosphate 6 buffer pH 7, 1% mercaptoethanol Rf 0.95(quenching) Analytical data Calculated C, 48.1; H. 4.58; N, 18.7 FoundC, 48.5; H, 5.9; N. 17.8

EXAMPLE lII Preparation of 5,1 l-Methenyltetrahydrohomofolic Acid 2.0 gof THHF was dissolved in 200 ml of formic acid and heated under argonfor 2 hours at C, cooled to room temperature and evaporated at reducedpressure at 30C. The residual oil was triturated in ethanol andrefrigerated overnight.

The suspension was stirred in ice for 30 minutes and was filteredthrough a Buchner Funnel. The filter cake was washed repeatedly withcold alcohol and evacuated on a high vacuum for 2 hours, followed bydrying overnight in vacuo.

uv-spectra, mp. Amin Amax )tmin Amax pH 1 266 289 298 324 pH 7 268 283297 329 pH 13 241 255 Tlc; cellulose, solvent system; 0.1 M phosphatebuffer pH 7, 1% mercaptoethanol Rf 0.60 (yellow fluorescence) Analyticaldata Calculated C, 44.37; H, 4.54; N, 16.49; Na, 7.73 Found I C 44.44;H, 5.02; N, 14.84; Na, 4,10

In all examples element analysis, u.v. (ultraviolet), and tlc (thinlayer chromatography) were preformed.

The embodiments of this invention in which an exclusive property orprivilege is claimed are defined as follows:

1. S-methyltetrahydrohomofolic acid and alkali metal salts thereof. I

2. 5,11-methylenetetrahydrohomofolic acid and alkali metal saltsthereof.

1. 5-methyltetrahydrohomofolic acid and alkali metal salts thereof.